Laforin-C329S is monomeric.

<p>(A) I<i>n vitro</i> dimerization assay. Recombinant proteins expressed in bacteria were purified and subjected to non-reducing, non-denaturing electrophoresis and were immunodetected using anti-laforin antibody. All mutants could form dimers with the exception of C329S (left panel). WT laforin and C329S were further analyzed by incubation in the presence of 10 mM DTT or 10 mM H₂O₂ and analyzed for the presence of monomeric and dimeric species (right panel). (B) Laforin C329S exclusion chromatography analysis. A single peak is observed in an elution volume corresponding to 32.5 kDa (molecular size markers are indicated). (C) <i>In vitro</i> phosphatase activity assay of the same sample showed that the C329S mutant displayed a catalytic activity comparable to the wild type protein. (D) <i>In vitro</i> dimerization assay of laforin-C329S expressed in mammalian cells. Crude lysates from HEK293 cells transfected with myc-laforin (wt, wild type) or myc-C329S plasmids were analyzed by Western blot. The electrophoresis was carried out in non-reducing, non-denaturing conditions, and the immunodetection was performed using anti-laforin antibody. The position of the monomeric and dimeric forms of laforin is indicated.</p

Human laforin contains nine cysteines.

<p>(A) Multiple sequence alignment of laforin orthologs. Sequences of several vertebrate (indicated by the brackets) and two invertebrate (<i>N. vectensis</i> and <i>T. gondii</i>) organisms were used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069523#pone.0069523.s001" target="_blank">Table S1</a> for details). The different cysteines are highlighted in yellow, and the position of cysteines in human laforin is marked (catalytic cysteine C266 appears in red). (B) Schematic of laforin domains (CBM: carbohydrate binding module; DSPD: dual-specificity phosphatase domain). The location of the nine cysteines is shown (blue numbers). (C) Tertiary structure prediction of the CBM (left) and DSPD (right) laforin domains. Homology models were created using the structures of glucoamylase (PDB: 1ACZ) and SEX4 (PDB: 3NME) as templates for the CBM and DSPD, respectively. The models were used to estimate the possible location of the cysteines in the tertiary structure. The cysteines studied in this work appear in yellow in the CBM and in orange in the DSPD; in grey, known tryptophans responsible of the carbohydrate binding.</p

Laforin-C329S and malin physically interact and form a functional complex in mammalian cells.

<p>(A) Yeast two-hybrid analysis. THY-AP4 yeast strain was transformed with pACT2-malin and LexA-laforin (WT or C329S) and the interaction was assessed by measuring the β-galactosidase activity. (B) Co-immunoprecipitation assay. HEK293 cells were co-transfected with plasmids myc-laforin (WT or C329S) and HA-malin. Cells were lysed and total lysates were incubated with anti-myc antibody and protein A/G beads. After washing, beads were boiled in loading buffer and purified proteins analyzed by SDS-PAGE and Western blot using anti-myc or anti-HA antibodies. (C) Ubiquitination analysis of R5/PTG by the laforin-malin complex. Overexpression of 6xHis-ubiquitin, pCMV-HA-malin, pCMV-myc-R5/PTG and pCMV-HA-laforin (wild type or C329S) in HEK293 cells, followed by lysis in presence of guanidinium chloride and purification of the ubiquitinated proteins by affinity chromatography using a cobalt resin. The result of the purification was analyzed using Western blot with anti-myc antibodies. Bound: proteins retained in the resin; crude extracts (50 µgr, C.E.) were immunodetected with anti-HA antibodies. *: polyubiquitinated forms.</p

Laforin-C329X does not dimerize, but maintains activity.

<p>(A) Laforin-C329X exclusion chromatography chromatogram. Recombinant laforin-C329X was purified via affinity chromatography and then subjected a HiLoad 16/60 Superdex 200 sizing column. A single peak elutes that corresponds to 32.5 kDA. (B) <i>In vitro</i> dimerization assay. Recombinant laforin-WT and laforin-C329X were purified via affinity chromatography, subjected to non-reducing, non-denaturing electrophoresis, and immunoblotted using anti-HIS antibody. (C) Co-sedimentation assay of protein and amylopectin (amylopectin binding assay). Recombinant histidine-tagged proteins were incubated with 5 mg/ml amylopectin, amylopectin was pelleted by ultracentrifugation, proteins in the pellet (P) and supernatant (S) were separated by SDS-PAGE, and visualized by Western analysis. Amylopectin-bound proteins are found in the pellet (P) and unbound proteins are found in the supernatant (S).VHR, <i>Vaccinia</i> virus phosphatase VH1-related; WT, laforin; C329X, laforin-C329X. (D) Recombinant laforin-WT, laforin-C329X, and laforin-C266S were purified and used for <i>in vitro</i> phosphatase assays employing the artificial substrate OMFP. The mutant C266S was used as a negative control. (E) Recombinant laforin-WT, laforin-C329X, and laforin-C266S were purified and used for <i>in vitro</i> phosphatase assays employing the phosphorylated glucan amylopectin. Inorganic phosphate release was detected via malachite green.</p